Abstract

A large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations. Unfortunately, little progress has been made in the treatment of patients with the most frequently observed driver oncogene, mutant KRAS. Furthermore, acquired resistance to the currently targetable driver mutations (EGFR mutant and ALK translocation positive tumors) is all but inevitable. We recently demonstrated that the basic helix-loop-helix transcription factor Twist1 cooperates with mutant Kras to induce lung adenocarcinoma in mouse models and that inhibition of Twist1 in murine models and human lung cancer cell lines led to oncogene induced senescence (OIS) and is some cases, apoptosis. Furthermore, we have found that TWIST1 is essential for tumor maintenance in human NSCLCs characterized by defined oncogenic drivers including KRAS mutation, EGFR mutation and c-MET amplification. As TWIST1 is not typically expressed post-natally, therapies directed against TWIST1 may be a more specific and perhaps less toxic therapy. Therefore, targeting the TWIST1 pathway represents an exciting and novel therapeutic strategy which may have a significant clinical impact.

Having identified TWIST1 as a prospective target, we employed a combined bioinformatic-chemical approach with in vitro and in vivo validation to identify pharmacologic inhibitors of TWIST1. We used gene expression profiles from several KRAS mutant human lung cancer cell lines following shRNA-mediated TWIST1 knockdown and from primary KrasG12D/Twist1 mouse tumors to perform connectivity map (CMAP) analysis, in an attempt to identify candidate agents that targeted TWIST1. We have validated the growth inhibitory effects of several of these agents in NSCLC cell lines. Furthermore, using a novel 3D organoid dissemination assay based on primary epithelial tissues from the Twist1 mouse, we demonstrated that several of our candidate agents produced dose-dependent inhibition of TWIST1 induced dissemination. Interestingly, the harmala alkaloid, harmine and several other harmala alkaloids ranked highly on the CMAP analysis. We have found that harmine could not only inhibit growth in several oncogene driver defined NSCLC cell lines through the induction of apoptosis but could also decrease TWIST1 levels through a post-transcriptional mechanism. Interestingly, the growth inhibitory effects of the harmala alkaloids correlated with the ability to degrade TWIST1. We are currently examining the in vivo efficacy of these agents using both xenograft mouse models as well as in our inducible KrasG12D/Twist1 model of lung adenocarcinoma. In conclusion, we have identified several putative inhibitors of TWIST1 through a CMAP analysis and demonstrated that treatment with harmala alkaloids leads to induction of apoptosis and degradation of TWIST1 in oncogene driven NSCLC. We have both in vitro and in vivo data suggesting that TWIST1 is not only essential for KRAS mutant NSCLC but more broadly for oncogene driven NSCLC. Therefore, these studies could lead to the development of a novel class of inhibitors which could have a significant clinical impact.